Mcl-1 expression is a predictive marker of response to gemcitabine plus nab-paclitaxel for metastatic pancreatic cancer

Antiapoptotic protein, including Mcl-1, expression is frequently observed in pancreatic cancer. Gemcitabine plus nabpaclitaxel (GnP) is the standard chemotherapy for metastatic pancreatic cancer (MPC); however, predictive markers for its efficacy remain unestablished. This study evaluated the association between GnP’s therapeutic effects and Mcl-1 expression in tissue samples obtained using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) for pancreatic tumor or percutaneous ultrasound-guided biopsy for metastatic liver tumor. We retrospectively reviewed 38 patients with histologically diagnosed MPC who received GnP as the first-line chemotherapy at our institute between December 2014 and July 2018. Post-immunohistochemistry analysis for Mcl-1 expression detection, patients were divided to into two groups based on the cell proportion showing Mcl-1 immunoreactivity: positive (> 20%; 23 [60.5%] patients) and negative (≤ 20%; 15 [39.5%] patients) groups. Clinical characteristics did not differ between the two groups. The Mcl-1 positive group showed a significantly higher disease control rate (95.7% vs. 73.3%; P = 0.046), longer progressionfree survival (PFS) (7.2 months vs. 4.9 months; P = 0.018) and longer overall survival (OS) (14.9 months vs. 9.2 months; P = 0.008) than the Mcl-1 negative group. Multivariate analysis showed that Mcl-1 expression was an independent predictive marker for PFS and OS. Mcl-1 expression could be a predictive marker for favorable response to GnP.


Acquisition of tissue samples
The specimens analyzed in this study were obtained using EUS-FNA or percutaneous ultrasound-guided liver tumor biopsy, which was performed for pathological diagnosis of PC before the initiation of chemotherapy.All EUS procedures were performed by endoscopists at our hospital using a linear-array echoendoscope (GF-UCT260; Olympus Medical Systems, Tokyo, Japan).EUS-FNA was performed with 19, 22, or 25-gauge franseen needles.The needle types, gauges and passes used were determined by the endoscopists.The aspirated biopsy materials were placed in a Petri dish with saline and into a formalin-filled container for histological analysis.Percutaneous ultrasound-guided liver tumor biopsy was performed using a 21-gauge aspiration needle (Sonopsy-C1; Hakko) or an 18-gauge core needle (MONOPTY).Thereafter, the formalin-fixed specimens were embedded in paraffin.Two experienced pathologists examined the hematoxylin and eosin-stained specimens and classified them as pancreatic ductal adenocarcinoma based on their findings.

Immunohistochemical analysis of the specimens
The formalin-fixed paraffin-embedded blocks of PC were cut into 3-5 μm slices, and the sections were subjected to immunohistochemical analyses to evaluate the immunoreactivity of the samples to Mcl-1.A monoclonal rabbit anti-Mcl-1 antibody (1:500, #39224; Cell Signaling Technology, Danvers, MA) was used for immunohistochemical staining.Antibody binding was visualized using the autoimmunostaining system VENTANA BenchMark ULTRA (Roche Diagnostics, Wetzlar, Switzerland) according to the manufacturer's instructions.
The investigator, who was blinded to the clinical data, assessed the percentage of stained cells.Specimens with more than 20% stained cancer cells in the cytoplasm, excluding non-cancer cells such as fibroblasts or immune cells, were categorized as Mcl-1 positive, according to previously published protocols (Fig. 1) [22][23][24] .

Statistical analysis
The χ 2 test and Fisher's exact test were used to compare categorical data, whereas the Wilcoxon rank sum text was used for the comparison of continuous and categorical variables.PFS and OS were estimated using Kaplan-Meier analysis, and the curves were compared using the log-rank test.Univariate and multivariate analyses were performed using the Cox regression method to evaluate the prognostic factors for PFS and OS.Variables with a P value < 0.20 in the univariate analysis were selected for inclusion into the multivariate analysis.Statistical analyses were performed using the JMP Pro 16 software (SAS Institute Inc., Cary, NC, USA).Statistical significance was set at P ≤ 0.05.

Patient characteristics and the outcomes of GnP therapy
A total of 38 patients with MPC were included in this study.All the patients were histologically diagnosed as having pancreatic ductal adenocarcinoma (EUS-FNA, 25 patients; percutaneous liver tumor biopsy, 13 patients).EUS-FNA was performed with 19-gauge needle in 1 patient, 22-gauge needle in 16 patients, and 25-gauge needle in 6 patients (detailed information of the needles was not available for 2 patients).There were no cases of cyst arising tumors such as invasive intraductal papillary mucinous neoplasm in imaging and histological diagnosis.The clinical characteristics of the patients are summarized in Table 1.The median age of the patients was 65 years (range, 38-80 years).Eighteen patients (47.4%) were male.Thirty (78.9%) and eight (21.1%)patients had an Eastern Cooperative Oncology Group PS of 0 or 1 and 2, respectively.The median body mass index of the patients was 21.1 kg/m 2 (range, 16.2-28.4kg/m 2 ).The primary tumor locations were the pancreas head in 12 patients (31.6%) and the pancreas body/tail in 26 patients (68.4%).Thirty-one patients (81.6%) had liver metastases, 8 patients (21.1%) patients had lung metastasis, and 15 patients (39.5%) had peritoneal metastasis.The median CEA, CA19-9, and albumin levels were 11 ng/mL (range, 1-338 ng/mL), 5484 U/mL (range, 2-100000 U/mL), and 3.6 g/dL (range, 2.4-4.5 g/dL), respectively.Fourteen patients (36.8%) had an NLR of less than 3. Biliary drainage was performed for eight patients (21.1%).

Association between Mcl-1 expression and outcomes of GnP therapy
Of the 38 patients included in this study, 23 (60.5%) tested positive for Mcl-1 expression and 15 (39.5%) tested negative.Table 1 shows a comparison of patient characteristics between the Mcl-1 positive and negative groups.There were no significant differences in baseline characteristics between the two groups.The patients in the Mcl-1 positive group showed a significantly higher disease control rate than those in the Mcl-1 negative group (95.7% vs. 73.3%;P = 0.046, Table 2).In addition, the PFS and OS of the patients in the Mcl-1 positive group were significantly longer than those of the patients in the Mcl-1 negative group (PFS: 7.2 months [range, 2.0-21.5 months] vs. 4.9 months [range, 2.0-7.8months], P = 0.018; OS: 14.9 months [range, 5.4-27.1 months] vs. 9.2 months [range, 3.0-13.3months], P = 0.008; Fig. 2).

Discussion
Expression of Mcl-1, an anti-apoptotic protein, is elevated in PC.However, the clinical significance of Mcl-1 expression in PC and its association with therapeutic response are unclear 25,26 .In the present study, the PFS and OS of the patients in the Mcl-1 positive group were significantly better than those of patients in the Mcl-1 negative group.Furthermore, multivariate analysis showed that Mcl-1 expression was an independent predictive marker for favorable PFS and OS.While it has been reported that PC with elevated Mcl-1 expression has a poor prognosis, Mcl-1 inhibitors induce apoptosis more strongly in tumors with elevated Mcl-1 19,27 .Gemcitabine has been reported to downregulate Mcl-1 expression and induce apoptosis in PC cells 28,29 .It is also reported that Mcl-1 is an important factor in determining the response to paclitaxel treatment, and the downregulation of Mcl-1 restores sensitivity to paclitaxel 30,31 .Together with these previous reports, favorable treatment responses of GnP in PC with elevated Mcl-1 expression may be possibly explained by downregulation caused by gemcitabine and subsequent restoration of sensitivity to paclitaxel.
GnP and fluorouracil-containing chemotherapeutic regimens (modified FOLFIRINOX and NALIRIFOX) are important treatment options for patients with MPC 32,33 .However, there are few clinical indicators of whether GnP or fluorouracil-containing regimens should be selected for chemotherapy.In the present study, patients with elevated Mcl-1 expression showed better treatment outcomes than those without elevated Mcl-1 expression.This finding suggests that Mcl-1 expression in tissue samples obtained by biopsy could be a predictive marker of the therapeutic effects of GnP.Studies conducted using comprehensive genomic profiling (CGP) tests have revealed that approximately 20% of patients with pancreatic ductal adenocarcinoma show mutations in homologous recombination genes, including BRCA1/2 and PALB2 34,35 .Moreover, patients with homologous recombination gene-mutated PC show favorable PFS and OS with platinum-containing chemotherapeutic regimens 35,36 .Mutations in homologous recombination genes are promising predictive markers of response to chemotherapy and can guide the selection of appropriate regimens.However, given that it takes a long time to obtain the results of a CGP test, it is difficult to factor in the presence of these mutations in the selection of chemotherapeutic regimens before initiating chemotherapy [37][38][39] .
The results of the present study demonstrated that Mcl-1 expression in specimens obtained using EUS-FNA or percutaneous liver tumor biopsy could predict the efficacy of chemotherapy.EUS-FNA is the primary method used for the pathological diagnosis of pancreatic tumor [40][41][42] .With the recent improvements in puncture needles, histological samples have also been used for CGP tests [43][44][45] .Percutaneous liver tumor biopsy also plays an important role in pathological diagnosis and tissue acquisition for genetic testing 46,47 .As immunohistological examinations are simple and rapid, Mcl-1 expression in specimens can be analyzed prior to the initiation of chemotherapy.Thus, tissue samples obtained by EUS-FNA or percutaneous liver tumor biopsy may contribute not only to the diagnosis of PC but also to the selection of chemotherapeutic regimens as well.This study has some limitations.First, this was a single-center retrospective study that examined cases meeting specific criteria, and it is unclear whether the findings can be generalized to patients with advanced PC who are undergoing GnP therapy in general.Second, we did not examine patients with MPC who received modified FOLFIRINOX as a first-line chemotherapeutic regimen, primarily because the number of patients who received the therapy was small.Further studies are needed to clarify the mechanism by which Mcl-1 influences the therapeutic effects of GnP.
In conclusion, this study demonstrated that Mcl-1 may be a predictive marker for the therapeutic effect of GnP in patients with MPC.This suggests that immunohistological examination of Mcl-1 expression before initiation of treatment could facilitate the selection of appropriate chemotherapeutic regimens.

Table 2 .
Treatment outcomes for patients with MPC who received GnP as first-line chemotherapy.Statistical significance was set at p < 0.05.Chi-square test, *Fisher's exact test.CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; NE, not evaluated.